This invention relates to growth hormone receptor and a novel growth hormone binding protein. The invention further relates to the synthesis of the growth hormone receptor and the growth hormone binding protein by recombinant means.
Human growth hormone (hGH) is a linear polypeptide with 191 amino acids and contains two intrachain disulfide bridges. The major biological effect of hGH is to promote growth. The organ systems affected include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys. Growth hormone exerts its action through interaction with specific receptors on cell membranes. Specific binding of growth hormone has been correlated with metabolic responses in primary cultures of adipocytes (Clemmons, D. R., et al., J. Clin. Invest. 106, 361-367[1981]; Gause, I., et al., Endocrinology 112, 1559-66[1983]). with developmental responses in a fibroblast cell line (Murphy, L. J., et al., Endocrinology 113, 750-57[1983]; Nixon, T. and Green, H. J. Cell. Physiol. 115, 291-96[1983]), and with growth responses in primary cultures of chondrocytes (Eden, S., et al., Endocrinology 112, 1127-29[1983]; Madsen, K., et al., Nature 304,545-47[1983]).
Several receptors have been cloned, for example EGF, insulin and I1-2. (See for example EP 0,192,392; EP 0,128,733; and, E.P. 0,162,699). The growth hormone receptor has been shown to be an integral membrane protein. Lectin-receptor interactions further suggest that growth hormone receptors contain a carbohydrate component associated with the extracellular domain of the receptor in conjunction with the growth hormone-binding site (Tsushima, T., at al., Biochem. J. 187, 479-92[1980]). Analysis of the growth hormone receptor by gel filtration has shown that the triton-solubilized receptor elutes with a M.sub.r of 200,000-300,000. (Waters, M. J. and Friesen, H. G., J. Biol. Chem. 254, 6815-25[1979]). It has been suggested that this technique probably overestimates the M.sub.r of the growth hormone receptor because it does not take into account the amount of Triton bound to receptor. (Hughes, J. P. et al. in Polypeptide Hormone Receptors, Posner, B. I. [ed.] [Dekker, M., N.Y.]) Recent studies have estimated the M.sub.r of the GH receptor to be in the range from 130,000 to 200,000 (rat hepatocytes, Donner, D. J. Biol Chem. 258, 2736-43[1983]; rat adipocytes, Carter-Su, C., et al., J. Biol. Chem., 259, 1099-104[1984]; and, IM-9 lymphocytes, Hughes, J. P., et al., Endocrinology 112, 1980-85[1983]). Under reducing conditions on SDS gel-electrophoresis, the M.sub.r of these receptors ranged from 108,000 to 112,000. In the absence of reducing agents, however, the GH receptor identified on rat hepatocytes migrated as a higher-M.sub.r species, suggesting that this GH receptor interacts via interchain disulfide bonds with other receptor or nonreceptor proteins (Donner, D., supra).
In contrast to the relatively high M.sub.r obtained for human and rat GH receptors in cross-linking studies, M.sub.r in the range of 50,000-67,000 have been obtained for the rabbit GH receptor in microsomal membranes (Tsushima, T., et al., FEBS Lett. 147,49-53[1982]). Additional data suggest that the GH receptor in intracellular membranes (microsomal membranes) is composed of noncovalently linked subunits, whereas the receptor on the cell surface (plasma membranes) is primarily composed of disulfide-linked subunits (Id.).
Several lines of evidence suggest the presence of two receptors in rabbit liver membranes capable of binding human growth hormone. One line of evidence is differences in binding observed when two or more GH tracers from different species are used to characterize GH receptors in the same rabbit liver preparation (Hughes, J. P., et al., Endocrinology 113,1904-6[1983]). Evidence in support of multiple classes of receptors also has been obtained from studies that have examined the binding characteristics/biological actions of cleaved forms of growth hormone (Maciag, T., et al., J. Biol. Chem. 255, 6064-70[1980]). Displacement studies using various growth hormones -rat, pig, human and bovine- and prolactin further suggest the presence of two growth hormone receptors. For example, the liver somatogenic receptor binds labelled growth hormone which label can be displaced by growth hormone but not prolactin. It was also observed that bovine or ovine prolactin can displace receptor bound human growth hormone (Waters, M. J. and Friesen, H. G., J. Biol. Chem. 254, 6815-25[1979]). Still other workers observed partial displacement by both ovine prolactin and bovine growth hormone, suggesting the presence of distinct somatogenic and lactogenic receptors, both of which bind human growth hormone. The liver may contain two types of growth hormone receptor, one with high affinity for rabbit growth hormone and relatively low affinity for human growth hormone (Hughes, J. P., Endocrinology 105, 414-20[1974]). Others suggest that the liver contains three types of receptors for hormones of the growth hormone-prolactin family, one specific for growth hormone, one specific for prolactin, and one that does not discriminate between the two (Wallis, M. et al. in Investigation of Membrane-Located Receptors eds. Reid, E et al. [Plenum Press, N.Y., 1984]).
Recently, data have accumulated suggesting the presence of a growth hormone binding protein in the serum of both humans and rabbits (Ymer, S. I. & Herrington, A. C., Mol. Cell. Endocrinol., 41, 153-161, [1985]; Herrington, A. C., et al., Proc. Annu. Meet. Endocr. Soc. Australia 28th. Adelaide, abstr. 77[1985]). Using monoclonal antibodies to the presumptive growth hormone receptor in rabbit and rat liver, there was observed a close antigenic and possible ontogenic relationship between membrane "receptor" and the cytosolic binding proteins found in the liver (Barnard, R. & Waters, M. J., J. Receptor Res. [1986]). Additional work has suggested that these cytosolic binding proteins may be newly synthesized serum binding proteins (Barnard, R. & Waters, M. J., Biochem J. 237, 885-892[1986]). The growth hormone binding protein, partially purified using hGH-affinity chromatography, was observed to be 74,000-85,000 mol, wt. and distinct from the major serum protein, human albumin (Ymer, S. I. and Herrington, A. C., Mol. and Cell. Endocr., 41:153-161 [1985]).
Many effects of growth are exerted through induction of somatomedins produced in the liver. Growth hormone binds to specific receptors on cell membranes, stimulating somatomedin generation. The net metabolic effects of growth hormone (and somatomedins) include stimulation of nucleic acid and protein synthesis, induction of positive nitrogen balance, stimulation of lipolysis, and a decrease in urea excretion. One variety of dwarfism referred to as Laron dwarfism occurs in patients in whom growth hormone is present but in whom receptors appear to be defective or absent so that somatomedin generation does not occur (Fishet et al., Isr. J. Med. Sci., 20:8-11 [1984] and Golde et al., N. Engl. J. Med., 303:1156-1159 [1980]).
Although the art has attempted to purify putative growth hormone receptors and binding proteins these preparations have been insufficiently pure to permit determination of the sequences of the growth hormone receptor and binding protein. This invention for the first time has established the precise identity of the growth hormone receptor and the ontogeny and identity of the growth hormone binding protein. It is an object of the present invention to purify the growth hormone receptor and the binding protein and DNA encoding same, and to produce useful quantities of each using recombinant DNA techniques. This and other objects of this invention will be apparent from the specification as a whole.